Promoter And RNA Interference Analysis Of Trilocular Gene Bjmc1 In Brassica Juncea L

It has been demonstrated that the multilocular trait of rapeseed is yield trait which is beneficial to the high yield of rapeseed.Bjmc1 gene is the multilocular gene in B.juncea.BjMc1 gene is the bilocular gene in B.juncea.Mutations in BjMc1 gene can cause three locules in the rapeseed silique with more seeds per silique and adding yield per plant.In this study,to find out the BjMc1 promoter core section,the promoter bioinformatics analysis and promoter truncation experiment were used.It provides the basis for the subsequent research on the transcription factors and the formation process of multilocular silique.This study also further verified the function of the Bjmc1 gene in the development of rapeseed silique by RNA interference with the expression of BjMc1.The results were described as follows:1.The BjMc1 promoter bioinformatics analysisOnline analysis of the upstream promoter sequence of BjMc1 gene with PlantCARE database found that the segment contained light responsive elements,elements involved in stress responsiveness,hormone responsive elements,the regulatory element involved in the MeJA-responsiveness?CGTCA-motif?,MYB binding sites,the elicitor-responsive element?ELI-box3?,regulatory elements related to meristem expression?CAT-box?,the cis-acting element conferring high transcription levels?5UTR Py-rich stretch?,and so on.2.The BjMc1 promoter core sectionThe BjMc1 promoter 5'terminal missing vectors were constructed in two times.They are P5K?-502bp?,P10K?-996bp?,P15K?-1568bp?,P20K?-2024bp?;P11K?-1118bp?,P12K?-1206bp?,P13K?-1314bp?,and P14K?-1414bp?.Histochemical staining analysis of GUS expression in the floral primordium on the inflorescence and seedling 12 d after germination of transgenic plants were performed.The results show that GUS gene driven by the deletions of P14K,P15K,and P20K expressed in the floral primordium,and the P5K,P10K,P11K,P12K,P13K,and PCK had no expression.Except P5K and PCK,the other vectors can drive the GUS gene expression in sepals,roots,stems,leaves and other tissues.Combining database analysis,it is speculated that the BjMc1 promoter core section locate at the upstream of-1414^-1300bp,controlling the BjMc1gene expression in the floral primordium,and CAT-box may be one of the core components.3.Homology analysis of promoter core sectionCompared with the Mc1 gene promoter core section 115bp in Brassica,it was found that the sequence was highly matched with the Mc1 gene promoter of other Brassica plants.It was 100%matched with BniMc1,96%matching with BraMc1 and BraMc1,and75%with AthMc1.This result suggested that the promoter core section 115bp was required for gene expression and highly conserved in Brassica species.The analysis of promoter sequence found that the five promoter regions contained the CAT-box meristem expression regulatory element,indicating that this element is the key regulatory element of Mc1 gene expression.4.The results of RNA interferenceBy analyzing the BjMc1 gene sequence and using the modified vector pC2300RNAi,the RNA interference vector pRNAi was successfully constructed,transferred to the homozygous bilocular piants and the positive transgenic plants were obtained.Phenotype observation and qRT-PCR analysis were performed for transgenic positive plants.The results indicate that there were three transgenic plants in T₀ generation that showed abnormal growth of stem apex.They are R67,R79,and R120.BjMc1 gene expression decreased in transgenic positive plants.Leaf becomes slender and leaf margin dentate reduced.And transgenic silique is generally shorter than the homozygous bilocular silique.There are some multrilocular-like siliques on the transgenic line.The multrilocular-like silique is similar with multrilocular silique,but only two chamber.In combination with the above results,the Bjmc1 gene can be considered to regulate the development of carpels in the Brassica juncea,and then determine the number of silique chamber.